(760c) Depletion of SIRT1, but Not SIRT2, Inhibits PMA-Stimulated Megakaryocytic Differentiation of the K562 Cell Line

Depletion of SIRT1, but not SIRT2, inhibits
PMA-stimulated megakaryocytic differentiation of the K562 cell line

Mark T. Duncan*, Zachary Mays, Nitin Kini, William
M. Miller

Department of Chemical and Biological
Engineering, Northwestern University, Evanston, IL, United States of America

*email: mtduncan@northwestern.edu

Sirtuins (SIRT1-7 in humans) are class III
NAD-dependent histone/protein deacetylases that have been implicated in
modulating the differentiation of many cell types including adipocytes and
endothelial, neural, and skeletal muscle cells.  Previously,
we have demonstrated that treatment with the pan-SIRT inhibitor Nicotinamide
(NIC) greatly enhances the polyploidization of human megakaryocytes (MKs)
derived from CD34+ cells in culture.  This effect was found to be, at least in
part, due to inhibition of SIRT1/2 deacetylases, as similar effects were
recapitulated by the SIRT1/2 specific inhibitor cambinol.  We are currently
using the cell line K562 to further investigate the roles of SIRT1/2 in MK
differentiation. Addition of phorbol 12-myristate 13-acetate (PMA)
induces K562 cells to express the MK antigen CD41 and exhibit morphological
characteristics of MK maturation, including enlargement of cell size,
polyploidization, and the appearance of cytoplasmic vacuoles.  Similar to primary cells, NIC treatment of differentiating
K562 cells led to an increased fraction of polyploid cells.  In addition, NIC
treatment reduced cell expansion and appeared to increase/accelerate apoptosis. 
Surprisingly, silencing of SIRT1 with lentiviral shRNAs strongly inhibited PMA-mediated
differentiation.  We consistently observed a moderate reduction in CD41 expression,
and a >50% reduction in polyploidization in SIRT1-silenced cells. SIRT2
silencing did not appear to affect differentiation.  Thus, we hypothesized that
SIRT1 may have an important role in PMA-induced cell signaling, and confirmed
its high expression in K562 cells via immunostaining.  Unexpectedly, SIRT1
appeared to be predominantly localized to the cytosol (both pre- and post-PMA
stimulation). We are currently investigating a possible role for cytosolic
SIRT1 in PMA-induced MAP kinase signaling, as a previous report suggests SIRT1 can
enhance ERK1/2 activation.¹  In addition, we have
begun to examine the effects of SIRT1 silencing on the differentiation of human
MKs derived from CD34+ cells in culture.  In an initial experiment, SIRT1
silencing reduced MK expansion, again suggesting SIRT1's importance in
regulating the cell cycle (though whether this is through a similar or distinct
mechanism from the K562 model cell line remains to be determined).  We are also
currently exploring knockdown of SIRT7, which is upregulated during primary Mk
differentiation. 

References

1          Huang,
J. et al. SIRT1 Overexpression Antagonizes Cellular Senescence with
Activated ERK/S6k1 Signaling in Human Diploid Fibroblasts. PLoS ONE 3,
e1710, doi:10.1371/journal.pone.0001710 (2008).